A Coregulatory Network of NR2F1 and microRNA-140 David Y. Chiang1,2☯, David W. Cuthbertson3☯, Fernanda R. Ruiz4, Na Li1*, Fred A. Pereira3,4* 1 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America, 2 Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, United States of America, 3 Bobby R. Alford Department of Otolaryngology- Head and Neck Surgery, Baylor College of Medicine, Houston, Texas, United States of America, 4 Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America Abstract Background: Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation. Results: Using a bioinformatic approach, we identified 11 potential miRNAs that might coregulate target genes with NR2F1 and analyzed their targets and potential roles in physiology and disease. We selected 6 miRNAs to analyze using quantitative real-time (qRT) -PCR and found that miR-140 is significantly down-regulated by 4.5-fold (P=0.004) in the inner ear of NR2F1 knockout (Nr2f1–/–) mice compared to wild-type littermates but is unchanged in the brain. Based on this, we performed chromatin-immunoprecipitation followed by qRT-PCR and confirmed that NR2F1 directly binds and regulates both miR-140 and Klf9 in vivo. Furthermore, we performed luciferase reporter assay and showed that miR-140 mimic directly regulates KLF9-3’UTR, thereby establishing and validating an example coregulatory network involving NR2F1, miR-140, and Klf9. Conclusions: We have described and experimentally validated a novel tissue-dependent coregulatory network for NR2F1, miR-140, and Klf9 in the inner ear and we propose the existence of many such coregulatory networks important for both inner ear development and function. Citation: Chiang DY, Cuthbertson DW, Ruiz FR, Li N, Pereira FA (2013) A Coregulatory Network of NR2F1 and microRNA-140. PLoS ONE 8(12): e83358. doi:10.1371/journal.pone.0083358 Editor: Bruce Riley, Texas A&M University, United States of America Received October 3, 2013; Accepted November 11, 2013; Published December 3, 2013 Copyright: © 2013 Chiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants T32AG00183 (to FRR) from the National Institutes of Health, 12BGIA12050207 (to NL) and 12PRE11700012 (to DYC) from the American Heart Association, and DC04585 and support from the Huffington Center on Aging and Department of Otolaryngology-HNS (to FAP). DYC was also supported by the Baylor College of Medicine Medical Scientist Training Program Caskey Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (FAP); [email protected] (NL) ☯ These authors contributed equally to this work. Introduction guidance, neocortical patterning, morphogenesis, and patterning of the cochlear sensory epithelium and the inner and Nuclear receptors are a large family of cellular regulators that outer hair cells in the organ of Corti [7–10]. A recent forward function as activators, repressors and silencers of transcription genetics screen in patients with congenital anomalies and when bound by specific ligands or cofactors [1,2]. They are cytogenetically balanced chromosomal rearrangements structured with a ligand-binding domain in the C-terminus, identified a critical paracentric microdeletion of the Nr2f1 locus, activation function domains, and a DNA-binding domain in the implicating haploinsufficiency of NR2F1 as a cause for a 4- N-terminus which binds to specific hormone response DNA year-old child’s deafness, dysmorphism and developmental elements in the genome [3]. Two major subclasses of nuclear delay [11]. receptors include the nuclear hormone receptors that respond In an effort to define transcription factor binding sites and to hormone ligands and the orphan nuclear receptors for which target genes in vivo, Montemayor et al. developed a endogenous ligands are not known [4]. Nuclear receptor methodology to identify NR2F1 target genes by intersecting subfamily 2 group F member 1 (NR2F1) is an orphan nuclear gene expression profiling, computational binding site queries, receptor required for the development of the inner ear and and evolutionary conservation data [12]. This approach is in cerebral cortex [5–8]. This is illustrated by loss-of-function in part based on the concept of ‘phylogenetic footprinting,’ which knockout mice (Nr2f1–/–) which impacts neurogenesis, axonal assumes evolution selects against mutations within DNA PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e83358 Coregulation by microRNAs and NR2F1 regions that have an important regulatory function; thus regenerating or rejuvenating hair cells and neurons in patients creating evolutionary cold spots within the critical genomic with partial or complete hearing loss [25,26]. This study segments that regulate gene expression [13]. Using this discovered coregulatory interactions between NR2F1 and approach we identified and validated several direct target miRNA-140 in their regulation of the gene Krüppel-like factor 9 genes of NR2F1, and began to uncover regulatory and (Klf9). This is the first description of such a pathway and feedback networks at the transcriptional and post-translational delineates the regulatory actions of a circuit involving the non- level that might be necessary in the development and steroidal nuclear receptor transcription factor and miRNAs. physiology of mammals. To date, relatively few coregulatory networks have been delineated for interactions between Materials and Methods nuclear receptors and microRNAs (miRNAs), and even fewer have been experimentally validated [14]. Ethics Statement MicroRNAs were discovered as small temporal RNAs that The Institutional Animal Care and Use Committee of Baylor regulate developmental transitions in C. elegans. They have College of Medicine approved all studies involving the use of diverse expressive patterns and regulate a variety of areas, animals as mandated by the United States federal government. including embryologic development, physiology, pathophysiology, and growth, development, progression, and Identification of the 11 miRNAs and their putative metastasis in cancer [15–17]. Currently, there are 2,578 targets (human) and 1908 (mouse) mature unique miRNAs identified In order to find the overlap of any miRNA gene with NR2F1 (mirBase 20, June 2013), and more than half are conserved in target genes, the human genome (GRCh37/hg19) and mouse vertebrates [18]. In humans, genes encoding miRNAs are genome (NCBI37/mm9) builds were viewed using the UCSC located in intergenic (52%), intragenic–intronic (43%), or genome browser [27] (http://genome.ucsc.edu) with the NR2F1 intragenic-exonic (5%) regions with both sense and antisense binding site custom track supplied by Montemayor et al. [12]. orientations [19]. This large family of 21-23 nucleotide single- The miRNA track was also viewed to identify every miRNA stranded non-coding RNAs negatively regulate gene mentioned by Wang et al. to be expressed in the developing expression at the post-transcriptional level by binding the mouse inner ear [22]. Other miRNAs were identified by 5’UTR, coding sequences, or 3’UTR. They have the ability to reviewing the literature involving ontogenesis or either promote degradation or suppress the translation of their NR2F1[21,22,28–30]. To find all possible gene targets for each target mRNA, depending on the complementarity with which miRNA, we utilized multiple database prediction tools: they bind [15,16]. These small non-coding RNAs are first miRBase (http://www.mirbase.org) [31], MicroCosm (http:// formed as pri-miRNAs which are capped, adenylated, spliced, www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5), and later cleaved by Drosha and Pasha endonucleases TargetScan 6.0 (http://www.targetscan.org), microRNA.org (RNase-III type) resulting in precursor miRNAs (pre-miRNA) (http://www.microrna.org/microrna/home.do), and PITA (http:// [16]. Pre-miRNAs are then processed by Dicer to form short genie.weizmann.ac.il/pubs/mir07/mir07_dyn_data.html) [32]. duplex RNAs, which are incorporated into a miRNA-induced The predicted genes from the different databases were silencing complex (miRISC), in order to interact with the target combined and redundant genes removed. The final lists were mRNA. compared with the putative NR2F1 targets identified by MicroRNAs have a specific role in the developing inner ear Montemayor et al. [12]. and hearing, where many miRNA expression profiles change both temporally and spatially during embryogenesis and post- natal maturation and are conserved through evolution [20–22]. miRNA binding site predictions The significance of miRNAs in audition is exemplified by MicroRNA.org (http://www.microrna.org/microrna/home.do)